Engineering ClpS for selective and enhanced N-terminal amino acid binding

Appl Microbiol Biotechnol. 2019 Mar;103(6):2621-2633. doi: 10.1007/s00253-019-09624-2. Epub 2019 Jan 24.


One of the central challenges in the development of single-molecule protein sequencing technologies is achieving high-fidelity sequential recognition and detection of specific amino acids that comprise the peptide sequence. An approach towards achieving this goal is to leverage naturally occurring proteins that function through recognition of amino (N)-terminal amino acids (NAAs). One such protein, the N-end rule pathway adaptor protein ClpS, natively recognizes NAAs on a peptide chain. The native ClpS protein has a high specificity albeit modest affinity for the amino acid Phe at the N-terminus but also recognizes the residues Trp, Tyr, and Leu at the N-terminal position. Here, we employed directed evolution methods to select for ClpS variants with enhanced affinity and selectivity for two NAAs (Phe and Trp). Using this approach, we identified two promising variants of the Agrobacterium tumefaciens ClpS protein with native residues 34-36 ProArgGlu mutated to ProMetSer and CysProSer. In vitro surface binding assays indicate that the ProMetSer variant has enhanced affinity for Phe at the N-terminus with sevenfold tighter binding relative to wild-type ClpS, and that the CysProSer variant binds selectively to Trp over Phe at the N-terminus while having a greater affinity for both Trp and Phe. Taken together, this work demonstrates the utility of engineering ClpS to make it more effective for potential use in peptide sequencing applications.

Keywords: Biosensor; N-end rule pathway; Peptide sequencing; Protein engineering.

MeSH terms

  • Agrobacterium tumefaciens / genetics
  • Agrobacterium tumefaciens / metabolism
  • Amino Acids / metabolism*
  • Carrier Proteins / genetics*
  • Carrier Proteins / metabolism*
  • Directed Molecular Evolution
  • Escherichia coli / genetics
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • Protein Binding
  • Protein Conformation
  • Protein Engineering*
  • Sequence Analysis, Protein*
  • Substrate Specificity


  • Amino Acids
  • Carrier Proteins
  • ClpS protein, E coli
  • Escherichia coli Proteins